Nucleic acid isolated from Tetrahymena which codes for a triterpenoid cyclase, its production, and use

ABSTRACT

The present invention relates to nucleic acids isolated from Tetrahymena which code for a ciliate-specific triterpenoid cyclase. The inventive nucleotide sequences and the polypeptide sequences derived therefrom demonstrate a surprisingly minimal sequence identity to known isoprenoid cyclases. The invention also relates to the use of nucleic acids for the regulation of triterpenoid cyclase expression in a host organism, as well as the targeted knockout or repriming of the triterpenoid cyclase gene. As a result of the altered expression of the triterpenoid cyclase, it is possible to modify and enrich the levels of multiple unsaturated fatty acids in the host organism.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a divisional of and claims, under 35 U.S.C. §120, the benefit of U.S. patent application Ser. No. 09/725,735, filed30 Nov. 2000, now U.S. Pat. No. 6,645,751 which is expresslyincorporated fully herein by reference.

FIELD OF THE INVENTION

The present invention relates to a triterpenoid cyclase (tetrahymanolcyclase) isolated from Tetrahymena, its coding nucleic acid, itsproduction, and use.

BACKGROUND OF THE INVENTION

The inventive triterpenoid cyclase catalyzes the formation oftetrahymanol from squalene by a direct cyclization of squalene (Capsi etal. (1968) J. AM. CHEM. SOC. 90:3563–3564; Abe et al. (1993) CHEM. REV.93:2189–2206). The triterpenoid cyclase also recognizes oxidosqualene asa substrate (Abe & Rohmer (1994) J. CHEM. SOC. PERKIN TRANS. 1:783–791). In addition to pentacyclic triterpenoids, the squalenetetrahymanol cyclase also catalyzes the formation of tetracyclictriterpenoids (Abe & Rohmer (1991) J. CHEM. SOC. CHEM. COMMUN. 902–903).Tetrahymanol (or gammaceran-3-ol), which is derived from isoprene, is amember of the isoprenoid class. Isoprenoids play an important role asphytohormones and carotenoids, and as components of chlorophyll,ubiquinone, plant resins, oils, and latex. As steroid hormones,isoprenoids effect important functions in animals. The formation oftetrahymanol can be reprimed in Tetrahymena by adding sterols, such ascholesterol (Conner et al. (1968) J. PROTOZOOL. 15:600–605; Conner etal. (1969) J. BIOL. CHEM. 244:2325–2333).

Isoprenoids are also important components of bacterial and eukaryoticmembranes. Similar to hopanoids and sterols (such as cholesterol),pentacyclic triterpenoid has tetrahymanol membrane-stabilizingproperties (Conner et al. (1968; 1969); Poralla et al. (1980) FEBS LETT.113:107–110). By restricting the fluidity of the lipid acid residues ofmembrane lipids, a condensed (membrane-solidifying) effect is achievedabove the phase transition temperature; while below the phase transitiontemperature, the fluidity of the membrane is increased, thus preventingthe optimal close packing of fatty acid residues. In addition, themembrane fluidity depends on the fatty acid composition of the membranelipids. The fluidity of membranes increases in proportion to the levelsof unsaturated fatty acids. With temperature changes, organisms are ableto regulate the fluidity of their membranes, for example, via the fattyacid composition. Below the phase transition temperature, isoprenoidsand unsaturated fatty acids increase the membrane fluidity via asynergistic effect. The inhibition of the synthesis of the cyclictriterpenoids alters membrane stability. This reduced membrane fluiditycan be compensated by an increased proportion of polyunsaturated fattyacids (PUFAs) in the membrane, i.e., the content of PUFAs can beincreased by inhibition of the triterpenoid cyclase.

The targeted modification of the composition of the fatty acid spectrumby means of gene technology for the commercial production of specialfatty acids or oils is described in Napier et al. (CURR. OPIN. PLANTBIOL. (1999) 2:123–127); Murphy & Piffanelli (SOC. EXP. BIOL. SEMIN.(1998) Ser. 57 (PLANT LIPID BIOSYNTHESIS) 95–130); and FACCIOTTI & KNAUF(In: ADV. PHOTOSYNTH. 6: LIPIDS IN PHOTOSYNTHESIS: STRUCTURE, FUNCTIONAND GENETICS. Siegenthaler & Murata (eds.) Kluwer Academic Publishers,Netherlands. (1998) 225–248). Thus, the modification of fatty acidcomposition can be regulated by altering the genes that code for enzymeswhich directly participate in the fatty acid synthesis, such asdesaturases. However, it has been reported that the level of PUFAs intransgenetic organisms was relatively low (Knutzon & Knauf (1998) SOC.EXP. BIOL. SEMIN. SER. 67:287–304).

The knockout or repriming of the gene that codes for triterpenoidcyclase and the resulting deficiency of tetrahymanol may influencemembrane fatty acid composition. However, the modified membrane fluiditycan be balanced by the production of unsaturated fatty acids.

Although the triterpenoid cyclase protein from Tetrahymena is known andhas been purified (Saar et al. (1991) BIOCHEM. BIOPHYS. ACTA,1075:93–101), it had not been possible to clone the gene fortriterpenoid cyclase from Tetrahymena (dissertation of Michal Perzl(1996) at the Faculty of Biology of Eberhard Karls University Tübingen).In previous studies, the gene sequence of triterpenoid cyclase could notbe determined by sequencing the purified protein, PCR with degenerativeprimers, or hybridization with heterologous probes.

The present invention relates to nucleic acids isolated from Tetrahymenawhich code for a ciliate-specific triterpenoid cyclase. The inventivenucleotide sequences and the polypeptide sequences derived therefromdemonstrate a surprisingly minimal sequence identity to known isoprenoidcyclases. The invention also relates to the use of nucleic acids for theregulation of triterpenoid cyclase expression in a host organism, aswell as the targeted knockout or repriming of the triterpenoid cyclasegene. As a result of the altered expression of the triterpenoid cyclase,it is possible to modify and enrich the levels of multiple unsaturatedfatty acids in the host organism.

SUMMARY OF THE INVENTION

The present invention is directed to an isolated nucleic acid comprisinga nucleic acid sequence encoding a polypeptide or functional variantthereof comprising the amino acid sequence of SEQ ID No. 12.

In a preferred embodiment, the isolated nucleic acid of the presentinvention comprises the nucleic acid sequences of SEQ ID No. 11 and SEQID No. 13. In another embodiment of the present invention, the isolatednucleic acid comprises at least 8 nucleotides of SEQ ID No. 11. Anotherembodiment is an isolated nucleic acid of the present invention whereinthe nucleic acid is selected from the group comprising DNA, RNA, anddouble-stranded DNA. In yet another embodiment of the invention, theisolated nucleic acid comprises one or more non-coding sequences.

In one aspect of the invention, the isolated nucleic acid is antisense.Another aspect relates to a vector comprising the isolated nucleic acidof the present invention, preferably the vector is an expression vector.In addition, the invention is also directed to isolated host cellscomprising said vector. Preferably, the host cells are protozoan, inparticular, ciliate.

Another embodiment is a method of producing the isolated nucleic acid ofthe present invention comprising the step of chemically synthesizingsaid nucleic acid. An additional embodiment is a method of producing theisolated nucleic acid comprising the step of isolating said nucleic acidfrom a gene library by screening said library with a probe.

The present invention also relates to an isolated polypeptide orfunctional variant thereof comprising the amino acid sequence of SEQ IDNo. 12. In particular, the invention relates to an isolated polypeptidecomprising at least 6 amino acids of SEQ ID No. 12.

Also within the scope of the present invention is a method of producinga polypeptide comprising culturing a host cell under conditionssufficient for the production of said polypeptide and recovering saidpolypeptide from the culture. The host cell may be a protozoa,preferably a ciliate.

One aspect of the present invention is directed to an antibody capableof binding the polypeptide of SEQ ID No. 12. Another aspect of thepresent invention relates to a method of producing said antibody ofcomprising the steps of immunizing a mammal with a polypeptide andisolating said antibodies.

In one embodiment of the present invention, the isolated nucleic acid isused to identify polypeptide variants comprising the steps of screeninga gene library with said nucleic acid and isolating said variant.

Also within the scope of the present invention is a method of enrichingthe squalene content, in a host cell comprising the step of inactivatingthe inventive nucleic acid. The nucleic acid may be inactivated by anantisense nucleic acid, by a deletion or insertion of a nucleic acidsequence, or mutation of said nucleic acid sequence. In particular, theinventive nucleic acid may be replaced with one or more selectablemarkers.

In another embodiment of the present invention, the isolated nucleicacid is used to produce cyclic triterpenoids, preferably pentacyclictriterpenoids, and most preferred tetrahymanol.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Results of a BLASTP database comparison of the protein sequenceaccording to SEQ ID No. 12 with protein databases.

FIG. 2A. Alignment of the protein sequence according to SEQ ID No. 12with known pentacyclic triterpenoid cylcases, lanosterol synthesisisolated from Alicyclobacillus acidoterrestris (SEQ ID No. 14).

FIG. 2B. Alignment of the protein sequence according to SEQ ID No. 12with known pentacyclic triterpenoid cyclases, squalene-hopene cyclaseisolated from Alicyclobacillus acidoterrestris (SEQ ID No. 15)

FIG. 3. Multiple alignment of the polypepetide sequence according to SEQID No. 12from Tetrahymena with known pentacyclic triterpenoid cyclases(SEQ ID Nos. 16–20).

FIG. 4. Schematic diagram of the gene structure of triterpenoid cyclasefrom Tetrahymena according to SEQ ID No. 11 and SEQ ID No. 9.

FIG. 5. Schematic diagram of the triterpenoid knockout construct. Aneo-cassette plasmid was inserted into the genomic sequence ofTetrahymena to produce the genetic knockout. The construct contains aneomycin resistance gene regulated by the Tetrahymena histone H4promoter and the 3′ flanked sequence of the BTU2 (β-tubulin 2) gene. Theneo-cassette plasmid was digested with EcoRV and Sma I. The resulting1.4 kb fragment was then ligated into the EcoRV-digested plasmid pgTHCproducing the plasmid pgTHC::neo.

FIG. 6. Schematic diagram of the pBTHC triterpenoid expressionconstruct. The plasmid (pBICH3-Nsi), which contains the non-coding,regulatory sequences of the Tetrahymena thermophila BTU1 (β-tubulin 1)gene with a Nsi I restriction site at the start codon, was used togenerate the tetrahymanol cyclase expression construct, pBTHC. Therestriction sites, Nsi I and BamHI, were added by PCR to the 5′ and 3′ends, respectively, of the coding sequence for Tetrahymena tetrahymanolcyclase. The PCR-modified tetrahymanol cyclase and the plasmidpBICH3-Nsi were digested with the restriction enzymes, Nsi I and BamHI,and purified by an agarose gel. The digested tetrahymanol cyclasefragment was then ligated into the plasmid pBICH3-Nsi and the resultingexpression vector, pBTHC, contained the complete coding sequence fortriterpenoid cyclase in the correct reading frame and the regulatorysequences of the BTU1 gene.

FIG. 7. Gamma-linolenic acid (GLA) in % of biodry mass of transformantscompared with wild type Tetrahymena. The GLA content of the Tetrahymenatriterpenoid cyclase knockout transformants was compared to Tetrahymenawild strain (B2086).

FIG. 8. Chemical structure of Tetrahymanol.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to nucleic acids isolated from Tetrahymenawhich code for a ciliate-specific triterpenoid cyclase. The inventivenucleotide sequences and the polypeptide sequences derived therefromdemonstrate a surprisingly low sequence identity to known isoprenoidcyclases. Another aspect of the invention is the genomic nucleotidesequence of the triterpenoid cyclase, which in addition to the codingsequence, contains non-coding nucleotide sequences, such as introns,promoters, and flanking sequences. The invention also relates to the useof nucleic acids for regulating expression, targeted knockout, orrepriming of this gene. By regulating the expression of the triterpenoidcyclase, it is possible to modify the levels of multiple unsaturatedfatty PUFAs in an organism. Moreover, as a result of the targetedknockout of triterpenoid cyclase, an enrichment of squalene, anintermediate in the synthesis of various triterpenoids, can be achieved.Squalene serves as a synthetic module for terpenes. Partially modifiedsqualene (e.g., in hydrogenated form) is used in dermatology productsand cosmetics, as well as in various derivatives found in skin and haircare products. In a further embodiment, a targeted overexpression of theinventive nucleic acids may result in the production of cyclictriterpenoids such as pentacyclic triterpenoids (e.g., tetrahymanol orhopane), and tetracyclic triterpenoids (e.g., lanosterol orcycloartenol). Cyclic triterpenoids are used in the synthesis of steroidhormones and saponins. These compounds display good skin penetration anddiffusion properties, and therefore, they are also used in cosmetics anddermatology products.

The inventive nucleic acids can be isolated from ciliates, preferablyTetrahymena, and most preferred from Tetrahymena thermophila.

One aspect of the present invention is to provide a nucleic acidisolated from Tetrahymena, which codes for a polypeptide with theactivity of a triterpenoid cyclase. Another aspect of the invention isthe regulation of gene expression or genetic knockout (adequateinhibition) of the triterpenoid cyclase in a host organism, preferablyin Tetrahymena, in order to increase the level of PUFAs produced in theorganism. In particular, a further aspect is to enrich the levels ofgamma-linolenic acid (GLA) by means of the host's own production of PUFAfor restoration of membrane stability.

A further embodiment of the present invention is the nucleic acidsaccording to SEQ ID No. 11 and SEQ ID No. 13, which code for atriterpenoid cyclase with the amino acid sequence according to SEQ IDNo. 12 or a functional variant thereof, and fragments thereof with atleast 8 nucleotides, preferably with at least 15 or 20 nucleotides, inparticular, with at least 100 nucleotides, and most preferred, with atleast 300 nucleotides (hereinafter called “inventive nucleic acid(s)”).The nucleic acid according to SEQ ID No. 11 represents theprotein-coding (or cDNA) sequence. The nucleic acid, according to SEQ IDNo. 13, represents a genomic sequence of the triterpenoid cyclase, whichin addition to the coding sequence, also contains non-coding nucleicacid sequences such as introns, promoters, and flanking sequences (suchas UTR) (FIG. 4).

The complete nucleic acid sequence according to SEQ ID No. 11 codes fora protein with 655 amino acids and a theoretical molecular mass of 76.02kDa. Sequence analysis, as provided in the present invention, confirmsthat the nucleic acid codes for the pentacyclic triterpenoid cyclaseisolated from Tetrahymena. A homology comparison enabled theidentification of the protein sequence as a triterpenoid cyclaseaccording to SEQ ID No. 12, which is derived from the nucleic acidsequence (SEQ ID No. 11). A BLASTP search (Altschul et al. (1997)NUCLEIC ACID RES. 25:3389–3402) was used for the homology comparison.Isopenoid cyclases (such as squalene-hopene cyclase, and lanosterol andcycloartenol synthetases) were identified as homologous proteins fromthe database (FIG. 1). The known triterpenoid cyclases (SEQ ID Nos.14–15) have a maximum identity of 28% as compared to the inventivepolypepetide sequence (FIG. 2). A multiple alignment of various knownisopenoid cyclases (SEQ ID Nos. 16–20) and the inventive polypeptidesequence is shown in FIG. 3. Homologies are observed in the conserveddomains. One such domain is the QW-motif (K/R X₂₋₃ F/Y/W L X₃ Q X₂₋₅ G XW (SEQ ID No. 21); Poralla et al. (1994) TRENDS IN BIOCHEM. SCI.19:157–158; Poralla (1994) BIOORG. MED. CHEM. LETT. 4:285–290), whichoccurs seven to eight times in squalene-hopene cyclases, and seven timesin oxidosqualene cyclases. The inventive polypeptide sequence has sevensuch QW-motifs, which are distinctly less conserved as compared to otherknown triterpenoid cyclases. Another conserved motif is theaspartate-rich motif (D V/L D D T A (SEQ ID No. 22); (Perzl et al.(1997) MICROBIOLOGY 143: 1235–1242), which is less conserved in theinventive polypeptide sequence in the homologous position (D T D D T G(SEQ ID No. 23)). Similar aspartate-rich motifs were found in otherenzymes of the isopenoid biosynthesis pathway (ASHBY ET AL. (1990) in:MOLECULAR BIOLOGY OF ATHEROSCHLEROSIS, 27–34. Ed. AD Attie, Amsterdam,Elsevier). While the inventive polypeptide sequence has been identifiedas triterpenoid cyclase, it differs considerably from other cyclases.The overall identity of the inventive polypeptide sequence as comparedto known cyclases is surprisingly small.

In a preferred embodiment, the inventive nucleic acid is a DNA or RNAmolecule, preferably a double-stranded DNA molecule, or a functionalvariant, of the nucleic acid sequence according to SEQ ID No. 11, or thegenomic nucleic acid sequence according to SEQ ID No. 13.

According to the present invention, the term “functional variant” isdefined as a nucleic acid which is functionally related to thetriterpenoid cyclase isolated from Tetrahymena, such as otherpentacyclic triterpenoid cyclases, or allelic or degenerative variants.

In a narrower sense, according to the present invention, the tern“variant” is defined as nucleic acids, which have a sequence identity ofapproximately 60%, preferably of approximately 75%, in particular, ofapproximately 90%, and most preferred, of approximately 95%. In thiscase, the degree of hybridization must also be taken into consideration.

The invention also comprises functional variants of the nucleic acid,which include nucleotide changes that do not alter the protein sequence.Due to the unusual codon use by ciliates (Wuitschick & Karrer (1999) J.EUKARYOT. MICROBIOL. 46(3):239–247), the described DNA sequence must bemodified for expression in other systems. For example, codons TAA andTAG, which code for glutamine in ciliates and are stop codons in mostother systems, are replaced with the codons CAA and CAG for expressionin other organisms. In addition, by modifying the sequence to therespective codon preference (or codon usage) of various organisms,protein expression can be optimized in these organisms. Modification ofnucleic acid sequences can be accomplished using methods well known toone skilled in the art. Alternatively, the sequence can be generated bychemically synthesizing oligonucleotides. The specific base changes inthe nucleic acid sequence can be determined utilizing known codon usagetables of the preferred expression systems (i.e., Codon usage tabulatedfrom the gene library: http://www.dna.affrc.go.jp/˜nakamura/CUTG.html).The present invention also comprises variants of nucleic acids.

The variants or fragments of the inventive nucleic acids can be used asprobes to identify additional functional variants, or as antisensenucleic acids. For example, a nucleic acid of at least approximately 8nucleotides is suitable as an antisense nucleic acid; a nucleic acid ofat least approximately 15 nucleotides as primer for PCR; a nucleic acidof at least approximately 20 nucleotides is suitable for identifyingother variants; and a nucleic acid of at least approximately 100nucleotides is may be used as a probe.

In a preferred embodiment, the protein-coding sequence of the inventivenucleic acid is deleted or replaced with a selectable gene, such as agene for antibiotic resistance. Using this approach, the native gene canbe “knocked out” in a host organism (gene knockout) by homologousrecombination. By replacing or deleting the triterpenoid cyclase gene,the synthesis of cyclic triterpenoids is inhibited resulting in amodification of the fatty acid composition in the host organism. Inanother aspect of the present invention, the inventive nucleic acidsequence is altered by a deletion, insertion, or point mutation leadingto either a reduced or an increased activity of triterpenoid cyclase.

In another embodiment, the triterpenoid cyclase isolated fromTetrahymena is expressed in another host cell or organism. To accomplishexpression in another host, the inventive nucleic acid is ligated into avector, preferably in an expression vector.

The expression vectors can be either prokaryotic or eukaryoticexpression vectors. For prokaryotic expression, the T7 expressionvector, pGM10, (Martin, 1996) can be expressed in E. coli cells. Thisvector contains an N-terminal “histidine tag” sequence (Met-Ala-His₆)which can be used for protein purification by Ni2+-NTA affinitychromatography. The eukaryotic expression vectors, p426Met25 andp426GAL1 (Mumberg et al. (1994) NUCL. ACIDS RES., 22:5767–68) aresuitable for the expression in Saccharomyces cerevisiae. In insectcells, Baculovirus vectors such as EP-BI-0127839 or EP-B1-0549721 may beused for protein expression, and for expression in mammalian cells, SV40vectors may be utilized. For detailed information concerning suitablevectors, refer to SAMBROOK ET AL. (1989) MOLECULAR CLONING: A LABORATORYMANUAL, Cold Spring Harbor Laboratory, Cold Spring, N.Y.; Goeddel, ed.(1990) METHODS IN ENZYMOLOGY 185 Academic Press; and PERBAL (1988) APRACTICAL GUIDE TO MOLECULAR CLONING, John Wiley and Sons, Inc. Therecombinant proteins or fragments thereof can be isolated by methods ofprotein purification that are well known to one skilled in the art(e.g., AUSUBEL ET AL. (1995), CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,Green Publishing Associates, New York).

The expression vectors preferably contain sequence elements whichinfluence expression, such as promoters, enhancer elements, and“upstream” activating sequences. Inducible and constitutive promoters,as well as tissue-specific promoters, are suitable for expression of theinventive nucleic acids. For example, the cauliflower mosaic virus(CaMV) 35S promoter (Restrepo et al. (1990) PLANT CELL 2:987–98) orpromoters which are activated during seed development, are suitable forthe expression in plant cells.

In a preferred embodiment of the present invention, the vectors used forexpression of the inventive nucleic acids are shuttle vectors (Wolk etal. (1984) PROC. NATL. ACAD. SCI. USA, 81:1561–1565; and Bustos et al.(1991) J. BACTERIOL. 174:7525–7533).

Generally, the expression vectors also contain regulatory sequenceswhich are compatible with the host cell, such as the trp promoter forthe expression in E. coli (refer to EP-B1-0154133), the ADR2 promoterfor the expression in yeast (Russel et al. (1983), J. BIOL. CHEM.258:2674–82), the Baculovirus polyhedrin promoter for the expression ininsect cells (refer to EP-B1-0127839), or the early SV40 promoter or LTRpromoters of MMTV (Mouse Mammary Tumor Virus; Lee et al. (1981) NATURE,294:228–32) for mammalian expression.

Vectors, which contain the inventive nucleic acid sequence, can betransferred to cells by infection, transfection, electroporation,particle bombardment, and other methods known to those skilled in theart. According to the present invention, transformation is generallydefined as the introduction of nucleic acids into a host cell (Sambrooket al. 1989; Potrykus (1991) ANNU. REV. PLANT PHYSIOL. PLANT MOL. BIOL.42:205–225; Christou (1993) CURR. OP. BIOTECH. 4:135–141).

In another aspect, the expression vector contains the inventive nucleicacids in functional combination with promoters or other regulatoryelements, or in combination with another gene. Preferably the additionalgene is a selection marker, such as a gene for antibiotic resistance. Ina preferred embodiment, the regulatory elements are nucleic acidsequences which are functionally active in ciliates, and in particular,active in Tetrahymena.

In another embodiment of the present invention, the inventive nucleicacid is expressed in Tetrahymena under the regulation of a strongpromoter, such as the Tetrahymena tubulin promoter (Gaertig et al.,(1999) NATURE BIOTECH. 17: 462–465). Preferably, the transformation ofTetrahymena may be achieved according to the methods described byGaertig et al. (1999); Gaertig & Gorovsky (1992) PROC. NATL. ACAD. SCI.USA 89:9196–9200. In another aspect, the regulatory elements forexpression may be the promoters for α- or β-tubulin isolated fromTetrahymena thermophila. The transformed Tetrahymena may be identifiedusing a selection media, such as a media that contains an antibiotic.

Overexpression of the inventive nucleic acids may result in theproduction of cyclic triterpenoids, preferably pentacyclictriterpenoids, and most preferred tetrahymanol.

The inventive nucleic acids can be chemically synthesized based on thesequences disclosed in SEQ ID No. 11 and 13, or based on the peptidesequence disclosed in SEQ ID No. 12 according to the phosphotriestermethod (Uhlman & Peyman (1990) CHEMICAL REVIEWS, 90:543, No. 4). Theinventive nucleic acids may also be isolated from an appropriate cDNA orgenomic library generated from an organism possessing isoprenoid cyclaseactivity (Sambrook, et al., 1989). Single-stranded DNA fragments derivedfrom the nucleic acid sequences according to SEQ ID No. 11 or 13 with alength of approximately 100–1000 nucleotides, preferably with a lengthof approximately 200–500 nucleotides, and most preferred, with a lengthof approximately 300–400 nucleotides may be suitable as probes to screena cDNA or genomic library.

Another embodiment of the present invention is a polypeptide with anamino acid sequence according to SEQ ID No. 12, or a functional variantthereof. Another aspect is amino acid fragments according to SEQ ID No.12 with at least six amino acids, preferably with at least 12 aminoacids, in particular, with at least 65 amino acids, and, most preferred,with at least 150 amino acids (hereinafter called “inventivepolypeptide”). In addition, polypeptides with a length of approximately6–12 amino acids, preferably approximately 8 amino acids, may contain anepitope. The epitope may be coupled with a carrier and then may be usedfor the production of polyclonal or monoclonal antibodies (see e.g.,U.S. Pat. No. 4,656,435). Polypeptides with a length of at leastapproximately 65 amino acids may also be used directly, without acarrier, to produce polyclonal or monoclonal antibodies.

Within the meaning of the present invention, the term “functionalvariant” is defined as a polypeptide which is functionally associatedwith the inventive peptide, i.e., it exhibits triterpenoid cyclaseactivity.

Also within the meaning of the present invention, the term “variant”includes polypeptides which have sequence homology. In particular,variant includes polypeptides with a sequence identity of approximately70%, preferably approximately 80%, in particular, approximately 90%, andmost preferred, approximately 95% compared to the protein with the aminoacid sequence according to SEQ ID No. 12.

In another aspect, the term variant includes deletions of thepolypeptide in the range of approximately 1–60 amino acids, preferablyapproximately 1–30 amino acids, in particular, approximately 1–15 aminoacids, and most preferred, approximately 1–5 amino acids. For example,methionine, the first amino acid of a protein, may be deleted withoutappreciably altering the function of the polypeptide.

Another embodiment of the present invention includes fusion proteins,which contain the above-described inventive polypeptides. The fusionproteins may possess triterpenoid cyclase activity, or may gain theactivity after the removing a portion of the fusion moiety. These fusionproteins contain, in particular, non-ciliated sequences of approximately1–200 amino acids, preferably approximately 1–150 amino acids, inparticular, approximately 1–100 amino acids, and most preferred,approximately 1–50 amino acids. Examples of non-ciliated peptidesequences include prokaryotic peptide sequences such as galactoidase ora histidine tag (i.e., Met-Ala-His₆ tag). The fusion protein containinga histidine tag is particularly advantageous for purifying the expressedprotein by affinity chromatography using metal ion-containing columnssuch as an Ni2+ NTA column (NTA: chelating nitrilotriacetic acid).

In a further aspect of the present invention includes variants of theinventive polypeptide which serve as epitopes that can be specificallyidentified by antibodies.

The inventive polypeptide can be produced, for example, by expressingthe inventive nucleic acid in a suitable expression system according tomethods which are generally known to a person skilled in the art.Strains of E. coli (DH5, HB101 or BL21), yeast (Saccharomycescerevisiae), insect cell lines (Lepidopteran species: Spodopterafrugiperda), or animal cells (COS, Vero, 293, and HeLa) are commerciallyavailable and suitable as host cells.

In another embodiment, peptide variants of the polypeptide can besynthesized by classical peptide synthesis methods (i.e., MerrifieldTechnique). These peptides can be used to produce antisera that can thenbe utilized to screen gene expression libraries as a means to identifyadditional functional variants of the inventive polypeptide.

Another aspect of the present invention relates to a method forproducing an inventive polypeptide by expressing an inventive nucleicacid in a suitable host cell, and if appropriate, isolating theinventive polypeptide.

The present invention also relates to antibodies which specificallyreact with the inventive polypeptide, in particular where variants ofthe polypeptide are either immunogenic or are rendered immunogenic bycoupling a suitable carrier, such as bovine serum albumin, to thevariant. The antibodies of the present invention may be eitherpolyclonal or monoclonal.

The method of producing antibodies is another aspect of the presentinvention. Antibodies can be generated according to methods generallyknown in the art. For example, a mammal, such as a rabbit, may beimmunized with the inventive polypeptide or variant thereof, and ifappropriate, in the presence of an adjuvant (e.g., Freund's adjuvant oraluminum hydroxide gels) (Diamond et al. (1981) N. ENGL. J. MED.,304:1344–49). The polyclonal antibodies produced in the animal as aresult of the immunological response, can easily be isolated from bloodusing methods generally known in the art. For example, antibodies may bepurified by column chromatography. A preferred method of antibodypurification is affinity chromatography, for example, the HiTrap™NHS-activated columns (Pharmacia, Piscataway, N.J.). Monoclonalantibodies can be produced according to the methods described by Winter& Milstein (Winter & Milstein, (1991) NATURE, 349:293–99).

In a preferred embodiment, the inventive nucleic acids are used toproduce a genetic knockout of triterpenoid cyclase in a host organism,preferably Tetrahymena. As a result of this gene knockout, the levels ofPUFA, in particular, GLA, are increased in the host organism. A geneknockout in Tetrahymena can be accomplished by homologous recombinationwherein a nucleic acid according to SEQ ID No. 11 or 13 is modified byreplacing the protein-coding sequence with a selectable gene (e.g., anantibiotic resistant gene) (Gaertig et al. (1994) NUCL. ACIDS RES.22:5391–5398; Kahn et al. (1993) PROC. NATL. ACAD. SCI. USA90:9295–9299). In addition to gene knockout technology (Galli-Taliadoros(1995) J. IMMUNOL. METHODS 181:1–15), an antisense strategy (Atkins etal. (1994) BIOL. CHEM. HOPPE SEYLER 375:721–729) or the antisenseribosome method (Sweeney et al. (1996) PROC. NATL. ACAD. SCI. USA93:8518–8523) may be utilized to reduce activity of triterpenoidcyclase.

The inventive nucleic acids of the present invention code for aciliate-specific triterpenoid cyclase isolated from Tetrahymena. Thesenucleic acids can be used to generate transgenic organisms, preferablyTetrahymena, which contain an increased level of unsaturated fattyacids. In a preferred embodiment of the present invention, the inventivenucleic acids may be utilized in the commercial production of PUFAs, inparticular, GLA. In another embodiment, the GLA content of thetransgenic organism (i.e., a transgenic Tetrahymena) may be increased bythe combination of a genetic knockout (or reduction) of tetrahymanolcyclase activity and a functional overexpression of fatty aciddesaturase.

In a preferred embodiment, a host organism, preferably Tetrahymena, istransformed with the inventive nucleic acid or the above-describedstructures (Gaertig et al. (1999); Gaertig & Gorovsky (1992); Gaertig etal. (1994); Kahn et al. (1993) PROC. NATL. ACAD. SCI. USA 90:9295–9299).

The transformed Tetrahymena may be grown and enriched in a selectivemedia, and then lipid(s) may be isolated from these cells according tostandard methods (e.g., Dahmer et al., (1989) J. AM. OIL CHEM. SOC.66:543). The methyl esters of fatty acids can be analyzed by gaschromatography.

The inventive nucleic acids or variants thereof can also be used toidentify related genes from other organisms, in particular, from otherprotozoa or protista, preferably ciliates (systematized according toCavalier Smith (1995) ARCH. PROTISTENK. 145:189–207). The inventivenucleic acid or variants thereof can be used as a labeled probe forisolating homologous genes. By hybridizing the labeled probe withisolated nucleic acids or other organisms, homologous nucleic acidsequences may be detected and isolated. The nucleic acid probe can belabeled in a manner known to the person skilled in the art (Ausubel etal., 1995; Sambrook et al., 1989). For example, radioactive nucleotidesor nucleotides linked to detectable molecules, such as fluorophores,digoxigenin, biotin, magnetic molecules or enzymes, may be used to labelthe nucleic acid probe. Homologous DNA sequences may be identified byhybridization of the labeled probe. Genomic or cDNA libraries may beused to screen for homologous sequences. In addition, Southern andNorthern blots may also be used to identify homologous sequences.Alternatively, homologous DNA may be hybridized with a labeled probe,and isolated by selective retention of the labeled probe (e.g., amagnet).

Homologous genes can also be isolated and cloned by means ofcross-hybridization, using methods which are known to a person skilledin the art, as described, for example, in Ausubel et al. (1995), orSambrook et al. (1989).

Oligonucleotides can be generated based on an isolated DNA sequencewhich represents the protein coding sequence and these oligonucleotidescan then be used to identify additional homologous nucleic acidsequences by polymerase chain reaction (PCR).

Detection with specific antibodies against the protein or variantsthereof (such as peptide antibodies) coded by the nucleic acid sequenceof the present invention, provides another option for isolatinghomologous proteins.

A plurality of well-established methods such as the methods described inSambrook et al. (1989) are well known in the art for isolating genomicDNA and mRNA, as well as for producing genomic and cDNA libraries.

EXAMPLES

The following examples serve to illustrate the invention, withoutlimiting the invention to these examples.

Example 1 Organisms and Culture Conditions

Tetrahymena thermophila (Strains B1868 VII, B2086 II, B*VI, CU522 wereprovided by Dr. J. Gaertig, University of Georgia, Athens, Ga., USA)were grown in modified SPP medium (2% proteosepeptone, 0.1% yeastextract, 0.2% glucose, and 0.003% Fe-EDTA (Gaertig et al. (1994) PROC.NATL. ACAD. SCI. USA 91:4549–4553)); skim milk medium (2% skim milkpowder, 0.5% yeast extract, 1% glucose, and 0.003% Fe-EDTA); or MYGmedium (2% skim milk powder, 0.1% yeast extract, 0.2% glucose, and0.003% Fe-EDTA) with the addition of an antibiotic solution (100 U/mlpenicillin, 100 μg/ml streptomycin, and 0.25 μg/ml amphotericin B) (SPPAmedium) at 30° C. in a 50 ml volume in a 250 ml Erlenmeyer flask, withshaking (150 rpm).

Plasmids and phages were reproduced and selected in E. coli XL1-BlueMRF′, TOP10F′, or JM109 cells. The bacteria were grown under standardconditions in LB or NZY medium with antibiotics in standardconcentrations (Sambrook et al., 1989).

Example 2 Preparation of a Tetrahymena Thermophila cDNA Library

Total RNA was isolated from Tetrahymena thermophila according to theguanidine thiocyanate/phenol/chloroform method (Chomzynski & Sacchi(1987) ANAL. BIOCHEM. 161:156–159). From total RNA, mRNA was extractedusing Oligotex™ mRNA Purification System (Qiagen, Germany). Thesynthesis of cDNA was performed according to the Stratagene ZAP Express®cDNA Synthesis and Cloning Kit (Stratagene, LaJolla, Calif.). Followingligation of EcoRI adapters and digestion with Xho I, the cDNA wasseparated on an agarose gel according to size (S: 500–1500 bp, B:greater than 1500 bp). The cDNA was purified from the gel (QIAquick™ GelExtraction Kit; Qiagen) and ligated into the ZAP express vector whichhad been cut with EcoRI and Xho I. The ligated cDNA was then packagedinto phage in vitro (Gigapack® III Gold; Stratagene). The phage werereproduced in E. coli XL1-Blue MRF′. The S-cDNA library containedapproximately 5×10⁵ clones with an average insertion size of 1.1 kb, andthe B-cDNA library contained approximately 6×10⁴ clones with an averageinsertion size of 2 kb.

Example 3 Preparing a Tetrahymena Thermophila Genomic DNA Library

Genomic DNA was isolated from Tetrahymena by the Urea Method (Gaertig etal., 1994). The genomic DNA was digested with EcoRI and the cut DNA wasthen ligated into an EcoRI-digested Lambda vector (ZapExpress,Stratagene). The library was generated using a method similar to themethod described for cDNA library preparation in Example 2.

Example 4 RT-PCR with Triterpenoid Cyclase-specific Primers

Sequence comparisons of known pentacyclic triterpenoid cyclases wereused to identify conserved regions. PCR primers were designed for thehighly conserved regions, GSWF/YGR/SWGV/I and DGGWGE, taking intoconsideration the ciliate codon or Tetrahymena codon usage (Wuitschick &Karrer (1999) J. EUKARYOT. MICROBIOL. 1999 46(3):239–47; CUTG, (CodonUsage Tabulated from gene library):http://www.dna.affrc.go.jp/˜nakamura/CUTG.html).

The following primers were used for the PCR reactions:

SEQ ID No. 1: 5′-GGTTCNTGGTAYGGTAGATGGG-3′; and SEQ ID No. 2:5′-TTCACCCCAACCACCATC-3′.

Isolated mRNA (100 ng) was used for the initial strand synthesiscatalyzed by the enzyme, AMV Reverse Transcriptase (Boehringer Mannheim,Indianapolis, Ind.). According to the manufacturer's protocol, the finalreaction volume was 20 μl and contained: 50 mM Tris-HCl (pH 8.5), 8 mMMgCl₂, 30 mM KCl, 1 mM DTT, 1 mM dNTPs, 2 U AMV Reverse Transcriptase,and 2 pmol Oligo-dT anchor primer, SEQ ID No. 3:5′-GACCACGCGTATCGATGTCGACT₁₆V-3′.

The reaction was incubated for 1 hour at 55° C., followed by a 10-minuteincubation at 65° C. An aliquot ( 1/10 volume) of the initial strandreaction was used for the PCR. The PCR reaction volume was 25 μl: 1%HotStarTaq™ PCR Buffer (Qiagen, Germany), 10 pmol of each gene-specificprimer (SEQ ID No. 1 and SEQ ID No. 2), 200 μM dNTPs, 1.5 mM MgCl₂, and1 U HotStarTaq™ DNA Polymerase (Qiagen). The PCR reaction was performedunder the following conditions: an initial denaturization at 95° C. for15 minutes, followed by 35 cycles at 94° C. for 30 seconds, 45° C. for30 seconds, 72° C. for 1 minute, and a final incubation at 72° C. for 10minutes. The PCR fragments were ligated into the TA Cloning® vector, pCR2.1, using the TA Cloning® kit (Invitrogen, San Diego, Calif.), and thentransformed into E. coli TOP10F′ (Invitrogen). Plasmid DNA was isolatedfrom positive clones (QIAprep® Spin kit, Qiagen) and sequenced.

Example 5 Isolation of the Triterpenoid Cyclase cDNA

Based on a preliminary sequence, new oligonucleotide primers weredesigned for PCR, SEQ ID No. 4: 5′-CTGTTGGAGCTGTTGTACCAGG-3′ and SEQ IDNo. 5: 5′-CGTAATTGACTCTTGCTAAACCTGG-3′.

The triterpenoid cyclase cDNA was generated by PCR using these primersin combination with vector-specific primers (T3 and T7). An aliquot ofthe S-cDNA library (2 μl; 10⁵ PFU/μl) was used for the PCR reaction (seeExample 2). PCR was performed according to the following protocol: DNAdenaturization for 15 minutes at 95° C.; followed by 35 cycles at 94° C.for 20 seconds, 57° C. for 20 seconds, 72° C. for 2 minutes; and a finalincubation at 72° C. for 10 minutes. The PCR products then were cloned(see Example 2) and sequenced.

A new primer was designed based on this sequence information. The primerwas located at the 5′-end of the cDNA sequence, SEQ ID No.6:5′-GCTAAAACTCTTTCATACATGAAGAAG-3′.

Using this primer in combination with a vector-specific primer, thecomplete cDNA was amplified by PCR (see above for PCR conditions) fromthe cDNA library. The PCR product was sequenced and the correspondingcDNA sequence is listed as SEQ ID No. 11. The protein sequence can bederived from the cDNA sequence taking into consideration the specificcodon usage.

Example 6 Isolation of the Genomic Sequence of Triterpenoid Cyclase

By screening a genomic library with a digoxigenin-labeled PCR-generatedtriterpenoid cyclase, a clone was isolated with a DNA insert ofapproximately 5000 bp. The clone was isolated from the phage by in vitroexcision producing the plasmid pgTHC. The DNA insert was sequenced (SEQID No. 13) by primer walking. Comparing the cDNA sequence (SEQ ID No.11) with this sequence, the introns and flanking sequences wereidentified (FIG. 4).

Example 7 Preparation of Triterpenoid Cyclase Knockout Constructs

A neo-cassette plasmid, p4T2-1ΔH3, (Gaertig et al., 1994) was insertedinto the genomic sequence of Tetrahymena to produce the geneticknockout. The construct contains a neomycin resistance gene regulated bythe Tetrahymena histone H4 promoter and the 3′ flanked sequence of theBTU2 (β-tubulin 2) gene. In Tetrahymena, this plasmid providesresistance to paromomycin. The plasmid p4T2-1ΔH3 was digested with EcoRVand Sma I. The resulting 1.4 kb fragment was then ligated into theEcoRV-digested plasmid pgTHC producing the plasmid pgTHC::neo. With asuccessful transformation, the gene for the triterpenoid cyclase wasreplaced by this construct by homologous recombination, and as a result,the cells were resistant to paromomycin.

Example 8 Preparation of the Expression Construct, pBTHC

The vector pBICH3 (Gaertig et al., 1999) contains the coding sequencefor the Ichthyophthirius I antigen (G1) preprotein flanked by thenon-coding, regulatory sequences of the Tetrahymena thermophila BTU1(β-tubulin 1) gene. A modified plasmid (pBICH3-Nsi) with a Nsi Irestriction site at the start codon (provided by J. Gaertig, Universityof Georgia, Athens, Ga., USA) was used to generate the tetrahymanolcyclase expression construct, pBTHC. The restriction sites, Nsi I andBamHI, were added by PCR to the 5′ and 3′ ends, respectively, of thecoding sequence for Tetrahymena tetrahymanol cyclase. Isolated plasmidcontaining the complete cDNA sequence for tetrahymanol cyclase (pTHC)was used as the template for PCR. The primers,

SEQ ID No. 7: 5;-CTCTTTCATACATGCATAAGATACTCATAGGC-3′ and SEQ ID No. 8:5′-GGCTTGGATCCTCAAATATTTTATTTTTATACAGG-3′,were used to generate the PCR products, which contained the completecoding sequence of tetrahymanol cyclase flanked by Nsi I and BamHIrestriction sites. The PCR products and the plasmid pBICH3-Nsi weredigested with the restriction enzymes, Nsi I and BamHI, and purified byan agarose gel. The resulting expression vector, pBTHC, contained thecomplete coding sequence for triterpenoid cyclase in the correct readingframe and the regulatory sequences of the BTU1 gene (FIG. 6). For thetransformation of Tetrahymena, the vectors were linearized by digestionwith the restriction enzymes, Xba I and Sal I.

In a successful transformation, these constructs replaced the BTU1 genevia homologous recombination, and as a result, the cells were resistantto Paclitaxel.

Example 9 Determining the Fatty Acid Spectrum of the Transformants

The fatty acid spectrum was determined using a gas chromatography systemwith a flame ionization detector (HP GC 6890; Hewlett-Packard,Wilmington, Del.). An FFAP (Free Fatty Acid Phase) Permbond (Macherey &Nagel GmbH, Düren) was used as the column. The fatty acids wereidentified by comparing the retention times of fatty acid methyl esterstandards. The concentration of fatty acids in the samples wasdetermined on the basis of the known standard concentration. Todetermine the fatty acid spectrum, isolated transformants in MYG medium(plus 10 μg/ml cholesterol) were grown at 30° C. with shaking (150) rpmfor 24–96 hours. An aliquot of the culture (50 ml) was centrifuged at1500×g for 15 minutes. The supernatant was discarded and the pellet wasfrozen at −80° C. and subsequently freeze-dried. The lyophilized sample(50 mg) was resuspended in 1 ml of 20% methanolic HCl and 1 ml ofmethanolic standard solution (1 mg/ml). To release the fatty acids andtheir transesterification of fatty acid methyl ester, the samples wereagitated in a water bath for two hours at 60° C., and then cooled toroom temperature. Aqueous, saturated sodium hydrogen carbonate solution(1 ml) was added to neutralize the sample, and the samples were mixedcarefully. The fatty acid methyl ester was extracted by the addition ofn-Hexane. The sample was thoroughly mixed, and a phase separation wasachieved by a 2-minute centrifugation at 4,300 rpm. About ⅔ of theupper, organic phase was removed, and 1 μl of the sample was injectedinto the GC column, and analyzed. The GLA content of the Tetrahymenatriterpenoid cyclase knockout transformants as compared to theTetrahymena wild strain (B2086) is shown in Table 1.

TABLE 1 Time % GLA/BTM % GLA/BTM % Difference % GLA/BTM % Difference(hours) B2086 AX004a AX004a AX081 AX081  6 1.10 1.03 −6.8% 1.08 −1.9% 211.24 1.32 +6.5% 1.44 +16.1% 32 1.37 1.57 +14.6% 1.76 +28.5% 54 1.54 1.96+27.3% 1.98 +28.6%

GLA content of the Tetrahymena triterpenoid cyclase knockouttransformants was compared to Tetrahymena wild strain (B2086). The tablespecifies the GLA percentile in the biodrymass (% GLA/BTM) and thepercentile difference (% difference) of the transformants compared tothe wild strain B2086.

Example 10 Macronucleus Transformation of Tetrahymena with theTetrahymanol Cyclase Expression Construct, pBTHC

Tetrahymena thermophila cells (5×10⁶; CU522) were used fortransformation. The cells were grown in 50 ml SPPA medium at 30° C. in a250 ml Erlenmeyer flask on a shaker (150 rpm) to a cell density ofapproximately 3–5×10⁵ cells/ml. The cells were pelleted for 5 minutes bycentrifugation (1200×g). The cell pellets were resuspended in 50 ml of10 mM Tris-HCl (pH 7.5), and then centrifuged as before. This washingstep was repeated. The cells were resuspended in 10 mM Tris-HCl (pH 7.5)with antibiotics at a cell density of 3×10⁵ cells/ml. The cells weretransferred to a 250 ml Erlenmeyer flask, and incubated for 16–20 hourswithout shaking at 30° C. (starvation phase). Following the starvationphase, the number of cells was determined. The cells were centrifuged asabove, and then resuspended in 10 mM Tris-HCl (pH 7.5) at aconcentration of 5×10⁵ cells/ml. One ml of cells was used for thetransformation. The transformation was performed by microparticlebombardment (see Example 12). To regenerate, the cells were resuspendedin SSPA medium, and incubated in an Erlenmeyer flask at 30° C. withoutshaking. After 3 hours, Paclitaxel was added to the medium in a finalconcentration of 20 μM. The cells were transferred in aliquots of 100 μlto 96-well microtiter plates and incubated in a humid, dark box at 30°C. After 2–3 days, the Paclitaxel-resistant clones were identified.Positive clones were hetero-inoculated in fresh medium with 25 μMPaclitaxel. By cultivating the cells with an increased Paclitaxelconcentration (up to 80 μM), a complete “phenotypic assortment” wasachieved as described by Gaertig & Kapler (1999).

To analyze the clones, DNA was extracted from approximately 4 mlcultures as described in Gaertig et al.(1994). DNA integrated in theBTU1 (β-tubulin 1) locus was amplified by PCR using BTU1-specificprimers SEQ ID No. 9: 5′-AAAAATAAAAAAGTTTGAAAAAAAACCTTC-3′, locatedapproximately 50 bp upstream of the start codon; and SEQ ID No. 10:5′-GTTTAGCTGACCGATTCAGTTGTTC-3′, 3 bp after the stop codon).

The PCR products was analyzed, uncut and cut with Hind III, Sac I or PstI, on a 1% agarose gel. The complete “phenotypic assortment” wasverified via RT-PCR with BTU1-specific primers (Gaertig & Kapler, 1999).

Example 11 Micronucleus and Macronucleus Transformation of Tetrahymenawith the Knockout Construct, pgTHC::neo

Tetrahymena strains of varying pairing types (CU428 VII and B2086 II)were separated in SPPA medium at 30° C. with shaking (150 rpm), andcultivated in an Erlenmeyer flask. With a cell density of 3–5×10⁵cells/ml, the cell were washed three times with 50 ml of 10 mM Tris-HCl(pH 7.5), and then resuspended in 50 ml of 10 mM Tris-HCl (pH 7.5)diluted with an antibiotic solution. The cells were incubated in anErlenmeyer flask at 30° C. without shaking. After approximately 4 hours,both cultures were recounted and diluted with 10 mM Tris-HCl (pH 7.5) to3–5×10⁵ cells and incubated for an additional 16–20 hours at 30° C.Following the starvation phase, the same (absolute) number of cells fromeach of the two cultures was mixed in a 2-liter Erlenmeyer flask. Thecells were incubated at 30° C. (start of conjugation) and after 2 hours,the efficiency of the conjugation was determined. To ensure a successfultransformation, approximately 30% of the cells should be in the form ofpairs.

For the micronucleus transformation, at 3, 3.5, 4, and 4.5 hourtimepoints following the start of conjugation, 1×10⁷ conjugated cells(5×10⁶ pairs) were centrifuged for 5 minutes at 1200×g. The cell pelletwas resuspended in 1 ml of 10 mM Tris-HCl (pH 7.5).

For transformation of the new macronucleus systems, 11 hours followingthe start of conjugation, the cells were centrifuged as above andsuspended in the Tris-HCl. The transformation was performed bymicroparticle bombardment (see Example 12). Cholesterol (10 μg/ml) wasadded to the medium for cultivating the tetrahymanol cyclase knockoutmutants.

The transformed cells were identified by paromomycin resistanceselection. During the transformation of the micronucleus, paromomycin(100 μg/ml final concentration) was added 11 hours after the start ofthe conjugation. The cells were distributed in aliquots of 100 μl onto96-well microtiter plates and then incubated in a moist box at 30° C.After 2–3 days, the paromomycin resistant clones were identified.Genuine micronucleus transformants could be differentiated by acomparison with the resistance for 6-methylpurin.

During the transformation of the macronucleus, paromomycin (100 μg/mlfinal concentration) was added approximately 4 hours following thetransformation. The cells were distributed in aliquots of 100 μl onto96-well microtiter plates and then incubated in a moist box at 30° C.After 2–3 days the resistant clones were identified. Positive cloneswere hetero-inoculated in fresh medium containing 120 μg/ml paromomycin.By culturing the cells in this high concentration of paromomycin, acomplete “phenotypic assortment” (Gaertig & Kapler, 1999) was achieved.

By crossing micronucleus transformants with a B*VI strain, homozygousknockout mutants were generated (Bruns & Cassidy-Hanley (1999) METHODSIN CELL BIOLOGY 62:229–240).

Example 12 Biolistic Transformation (Microparticle Bombardment)

The transformation of Tetrahymena thermophila is achieved by the methodsdescribed by Bruns & Cassidy-Hanley (1999) METHODS IN CELL BIOLOGY62:501–512; Gaertig et al. (1999); and Cassidy-Hanley et al.(1997)GENETICS 146:135–147. The use of the Biolistic® PDS-1000/He ParticleDelivery System (BIO-RAD) is detailed in the manufacturer's manual.

For the transformation, gold particles (6 mg; 0.6 μm; BIO-RAD) wereloaded with linearized plasmid DNA (10 μg) as described by Sanford etal. (1991) BIOTECHNIQUES 3:3–16; and Bruns & Cassidy-Hanley (1999).

Preparation of the gold particles. The gold particles were resuspendedin 1 ml ethanol and then thoroughly vortexed 3 times for 1–2 minutes.Subsequently, the particles were centrifuged for 1 minute at 10,000×g,and the supernatant was carefully removed with a pipet. The goldparticles were then resuspended in 1 ml sterile water and centrifuged asdescribed above. This wash phase was repeated. The particles were thenresuspended in 1 ml 50% glycerol and stored in aliquots of 100 μl at−20° C.

Loading the gold particles with DNA. All preparation was performed at 4°C. The gold particles, DNA vector, 2.5 M CaCl₂, 1 M spermidine, 70% and100% ethanol were cooled on ice. The linearized DNA vector (10 μl; 1μg/ml) was added to the gold particles (100 μl) and carefully vortexedfor 10 seconds. Initially, 2.5 M CaCl₂ (100 μl) was added to theDNA-gold particles, vortexed for 10 seconds, and then spermidine (40 μl)was added and the sample was carefully vortexed for 10 minutes.Following the addition of 70% ethanol (200 μl), the DNA-gold particleswere vortexed for 1 minute and then centrifuged for 1 minute at10,000×g. The pellets were resuspended in 100% ethanol (20 μl),centrifuged, and then resuspended in 35 μl 100% ethanol.

Preparation of the macrocarrier. The macrocarrier holder, macrocarrier,and stop screens were placed in 100% ethanol for several hours, and therupture disks were placed in isopropanol. Subsequently, one macrocarrierwas inserted into the macrocarrier and air-dried. The prepared goldparticles were then placed carefully in the center of the macrocarrierusing a pipet. The macrocarrier was stored in a box with hygroscopicsilica gel until the transformation.

Transformation. Prepared cells (1 ml) were placed in the center of acircular filter which was situated in a Petri dish. The filter wasmoistened with 10 mM Tris-HCl (pH 7.5), and placed into the lowestinsert slot of the transformation chamber of the Biolistic® PDS-100/HeParticle Delivery System. Transformation with the prepared goldparticles was performed at a pressure of 900 psi (two 450 psi rapturedisks) and a vacuum of 27 mmHg in the transformation chamber. The cellswere then transferred immediately to an Erlenmeyer flask with 50 ml SPPAmedium and incubated at 30° C., without shaking.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

The disclosures of all references and publications cited above areexpressly incorporated by reference in their entireties to the sameextent as if each were incorporated by reference individually.

1. A method of enriching the unsaturated fatty acid content in a hostcell comprising the step of inactivating the transcription of a nucleicacid encoding a polypeptide comprising the amino acid sequence of SEQ IDNO: 12; in said host cell.
 2. The method of claim 1, wherein saidnucleic acid is inactivated by an antisense nucleic acid specific forsaid nucleic acid.
 3. The method of claim 1, wherein said nucleic acidis inactivated by a method selected from the group consisting ofdeletion of said nucleic acid, insertion of a nucleic acid, and mutationof said nucleic acid.
 4. The method of claim 3, wherein said nucleicacid is replaced with one or more selectable markers.
 5. The method ofclaim 1, wherein the squalene content in the host cell in enriched.